Obesity is associated with reduced adenosine triphosphate (ATP) turnover in skeletal muscle. This condition results in unfavorable outcomes, proposed to range from decreased capacity for physical activity, to impaired activation of physiological mechanisms associated with cell function, to increased oxidative stress and the development of insulin resistance. The abundance of the beta subunit of the ATP synthase ( -F1-ATPase) in muscle mitochondria is decreased in obese, insulin-resistant individuals. -F1- ATPase makes up the catalytic site of the ATP synthase, and it is a rate-limiting component of ATP synthesis. We propose that muscle -F1-ATPase synthesis is reduced in obesity. Because measurement of stable isotopic enrichment of individual proteins is not practical using traditional gas chromatography-mass spectrometry approaches for proteins that are found in small amounts in skeletal muscle, we have developed an approach to quantify the isotopic enrichment of in vivo labeled muscle -F1-ATPase using HPLC-ESI-MS/MS. It is based on the quantification of the isotopic enrichment of a unique peptide of muscle -F1-ATPase. Using this approach we will test the hypothesis that the rate of muscle -F1-ATPase synthesis is reduced in obese individuals. We also intend to investigate the effects of increased plasma amino acid concentrations as well as exercise on stimulating the synthesis rate of -F1-ATPase in skeletal muscle of both obese and non-obese individuals. Muscle -F1-ATPase synthesis, which is the main end-point of this proposal, will be determined using an intravenous constant infusion of d9-leucine in obese and non-obese subjects, and by measuring the d9-leucine enrichment of a -F1-ATPase peptide. The following conditions will be tested: saline infusion (control), amino acid infusion, aerobic exercise, and a combination of aerobic exercise with amino acid infusion. These studies will for the first time determinate the rate of synthesis of muscle -F1-ATPase in humans in vivo, and how it is altered by interventions known to promote muscle protein anabolism. Overall, the results of these studies will lead to better understanding of the mechanisms regulating the abundance of -F1-ATPase in skeletal muscle in both obese and non-obese individuals. Further, they will provide scientific knowledge to base lifestyle interventions to improve muscle ATP turnover in obese individuals. PUBLIC HEALTH RELEVANCE: Obesity is associated with reduced adenosine triphosphate (ATP) turnover in skeletal muscle, a condition that can impair muscle metabolism. The proposed research will discover mechanisms responsible for decreased content in the protein -F1-ATPase, which is directly responsible for ATP assembly in skeletal muscle, and also examine the effectiveness of interventions to increase the rate of production of -F1-ATPase in skeletal muscle. This is important in order to develop appropriate interventions to improve muscle metabolism in obese individuals.